Methods for enhancing graft survival by modulating heme oxygenase activity

ABSTRACT

Methods are provided wherein the survival of an organ transplant is enhanced by introducing into cells of the transplant a nucleic acid molecule that modulates heme oxygenase-I activity therein. Nucleic acid molecules that modulate heme oxygenase-I activity and therefore find use in the described methods include, for example, molecules that encode a polypeptide that itself exhibits heme oxygenase-I activity or antisense oligonucleotides that act to inhibit heme oxygenase-I activity in a cell.

This application is a continuation-in-part of application Ser. No.09/216,005, filed Dec. 17, 1998, now abandoned and is a continuation ofInternational Application No. PCT US99/30089, filed Dec. 17, 1999.

BACKGROUND OF THE INVENTION

The inflammatory process is an extraordinarily complex process, varyingwith the cause of inflammation, the site of the inflammation, and thenature of the insult. Numerous different types of leukocytes areattracted to the site where the inflammatory process is initiated. Thedifferent leukocytes initiate different biological processes to respondto the insult. While in many situations, the inflammatory response ishealthy in destroying a pathogen, in other situations, such asautoimmune diseases and transplantation, the inflammatory response isundesirable. In the latter case, this leads to rejection and loss of theimplanted organ, which in most cases will be fatal.

A number of different avenues have been investigated to encourage theretention of allografts. For the most part, these avenues have involvedgeneral immunosuppression, using drugs such as cyclosporin and FK506.Extensive efforts have been directed to inducing anergy toward theforeign tissue. Also, the role of various factors has been investigated,where by modulating the level of those factors, the immune response maybe diminished. For the most part, the primary approach has been the useof drugs which suppress the entire immune system, therefore leaving thepatient vulnerable to adventitious infection.

Because of the restricted availability of donor organs, considerationhas been given to using xenografts for temporary maintenance, while anacceptable allogenic organ is identified. The xenografts not only differas to the MHC, but will also have numerous other epitopes differing fromthe host. Therefore, additional rejection mechanisms are brought to bearagainst the xenograft.

Heme oxygenases (HO) are the rate-limiting enzymes that catalyze theconversion of heme to biliverdin, carbon monoxide (CO) and free iron,the first step in the oxidative conversion of heme to bilirubin.Recently, great interest has been placed on the role of heme oxygenasein cellular responses to stress and insult, including ischemic andimmunogenic effects. All of the end products of heme degradation,including biliverdin, bilirubin, and CO, are known to modulate immuneeffector functions. Biliverdin has also been shown to inhibit humancomplement in vitro. Bilirubin inhibits human lymphocyte responses,including PHA-induced proliferation, IL-2 production, andantibody-dependent and -independent cell-mediated cytotoxicity. Inaddition, heme oxygenase-I (HO-I) upregulation correlates with increasedproduction of nitric oxide (NO), an important effector molecule involvedin inflammation and immune regulation. On the other hand, NO is alsoknown to induce HO-I expression, while CO directly inhibits nitric oxidesynthase (NOS) activity by binding to the heme moiety of the NOS enzymeand thus downregulating NO production. Like NO, CO contributes toendothelium-dependent vasodilation and inhibits platelet aggregation byelevating intracellular cGMP levels. The deleterious effects ofhyperoxia are thought to be mediated by reactive oxygen species. Bothbiliverdin and bilirubin are efficient peroxyl radical scavengers thatinhibit lipid peroxidation. Bilirubin scavenges peroxyl radicals asefficiently as α-tocopherol, which is regarded as the most potentantioxidant of lipid peroxidation. On the other hand, oxygen radicalsmay trigger cascade of antiapoptotic events, including those thatinvolve activation of bcl-2 protooncogene. All these factors point to acomplex picture of putative regulatory interactions between the HOsystem and the host cytokine network set in motion through thebiological activity of heme degradation products.

There is a pressing need to find alternative modalities which willenhance and extend transplant survival. These modalities may find use inconjunction with other drugs, where lower levels of other drugs havingsignificant side effects may be used effectively, so as to reduce thedetrimental side effects. Thus, there is substantial interest indeveloping new approaches to improving transplant outcome, where a drugmay act by itself or in conjunction with other drugs.

BRIEF DESCRIPTION OF THE RELEVANT LITERATURE

Heme oxygenase has been the subject of numerous studies as evidenced bythe review article, Abraham et al., Int. J. Biochem. 20(6):543-558(1988). Recently, modulation of heme oxygenase activity has beendescribed in Raju and Maines, Biochimica et Biophysica Acta 1217:273-280(1994); Neil et al., J. of Ocular Pharmacology and Therapeutics11(3):455-468 (1995); Haga et al., ibid. 1316:29-34 (1996); Willis etal., Nature Medicine 2(1):87-90 (1996); and Agarwal et al.,Transplantation 61(1):93-98 (1996).

SUMMARY OF THE INVENTION

In one embodiment of the present invention, methods are provided forextending the survival of an organ transplant in a recipient, whereinthose methods comprise contacting the organ transplant with a nucleicacid that functions to modulate heme oxygenase-I activity in thosecells, whereby the survival time of the organ transplant in therecipient is extended. In one embodiment, the nucleic acid encodes aheme oxygenase polypeptide.

Yet another embodiment of the present invention is directed to methodsfor extending the survival of an organ transplant in a recipient,wherein the methods comprise contacting cells of the organ transplantwith a nucleic acid encoding a polypeptide having heme oxygenaseactivity, wherein the nucleic acid is expressed in the cells in anamount sufficient to increase heme oxygenase activity therein, wherebythe survival time of the transplant is extended. Additional embodimentswill become evident upon a reading of the present specification.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph of the inhibition of target cell lysis by treatmentwith metalloprotoporphyrins. T-cell mediated cell lysis was evaluated inthe presence of varying amounts of Zn- and Co-protoporphyrin in a fourhour chromium release assay.

FIG. 2 is a graph showing the prolongation of heart allograft survivalfollowing metalloprotoporphyrin therapy. CBA recipients of C57Bl/6 heartallografts were either untreated or treated as follows:Zn-protoporphyrin group (n=4); Zn-protoporphyrin was administered at 10mg/kg/day on day −1 before transplantation and on days 1-9 posttransplantation. Co-protoporphyrin group (n=4); Co-protoporphyrin wasadministered at 20 mg/kg/day on days 0-5 post-transplantation;Zn-protoporphyrin pretreatment group (n=3); heart donors were treatedone day before transplantation with 50 mg/kg Zn-protoporphyrin.

FIG. 3 shows the nucleic acid sequence (SEQ ID NO:1) of a cDNA encodinghuman heme oxygenase-I (nucleotides 81-944).

FIG. 4 shows results demonstrating prolongation of liver isograftsurvival. Lean Zucker rats served as recipients of liver transplantsfrom obese Zucker donors. Donor rats were either pretreated with CoPP orAd-HO-I or remained untreated before liver procurement followed by 4hours of cold ischemia. Control animal survival at 14 days was 40%versus 80% and 81.8% in the CoPP and the Ad-HO-I group, respectively(n=10-11 rats/group).

FIG. 5 shows bile production in fatty livers perfused for 2 hours on theisolated perfusion rat liver apparatus after 6 hours of cold ischemia.Animals were pretreated with metalloporphyrins, or with Ad-HO-I genetransfer, or left untreated. Bile production at 30-minute intervalsthroughout the reperfusion period was significantly higher in theCoPP/Ad-HO-I groups (*P<0.05) as compared with untreated, ZnPP-, or Ad-βGal-pretreated controls. These data represent the mean±SE of 4-10independent perfusions for each group. *P<0.05 versusuntreated/ZnPP-treated controls.

DESCRIPTION OF THE SPECIFIC EMBODIMENTS

Methods are herein provided for prolonging the survival of transplantsin a mammalian host. In a preferred embodiment, the methods comprisecontacting cells of the organ transplant with a nucleic acid moleculethat functions to modulate heme oxygenase-I (HO-I) activity in cells ofthe organ transplant, whereby the survival time of the organ transplantin the recipient is extended. For the most part, nucleic acid moleculesthat function to modulate HO-I activity in cells will be nucleic acidmolecules that encode a polypeptide that exhibits at least onebiological activity that is normally associated with the human HO-Ipolypeptide encoded by nucleotides 81-944 of the nucleic acid shown inFIG. 3 (SEQ ID NO:1) or will be antisense oligonucleotides whosesequences are derived from and/or based upon nucleotides 81-944 of thehuman heme oxygenase-I nucleotide sequence shown in FIG. 3 (SEQ ID NO:1)or non-coding sequences of a heme oxygenase-encoding nucleic acidmolecule.

By “heme oxygenase-I”, “HO-I” and grammatical equivalents thereof ismeant the polypeptide encoded by nucleotides 81-944 of the nucleotidesequence shown in FIG. 3 (SEQ ID NO:1) and homologs thereof whichexhibit at least one biological activity that is normally associatedwith the human heme oxygenase-I enzyme. Preferably, the heme oxygenase-Iactivity exhibited by the potypeptides is the ability to catalyze thefirst step in the oxidative degradation of heme to bilirubin (Tenhunenet al., J. Biol. Chem. 244:6388-6394 (1969) and Tenhunen et al., J. Lab.Clin. Med. 75:410-421 (1970)). In this regard, Applicants note thatquick, easy and reliable assays are known in the art to determinewhether a polypeptide exhibits heme oxygenase-I activity, wherein thoseassays may be routinely employed to test the ability of any polypeptidefor the presence of heme oxygenase-I activity. For example, theproduction of bilirubin from heme can be determined using aspectrophotometer, whereby the increase in optical density at 468 mμ ina mixture of the peptide, hemin, biliverdin reductase and NADPHindicates heme oxygenase activity.

The terms “polypeptide” and “protein” may be used interchangeablythroughout this application and mean at least two covalently attachedamino acids, which includes proteins, polypeptides, oligopeptides andpeptides. The protein may be made up of naturally occurring amino acidsand peptide bonds, or synthetic peptidomimetic structures. Thus “aminoacid”, or “peptide residue”, as used herein means both naturallyoccurring and synthetic amino acids. For example, homo-phenylalanine,citrulline and noreleucine are considered amino acids for the purposesof the invention. “Amino acid” also includes imino acid residues such asproline and hydroxyproline. The side chains may be in either the (R) orthe (S) configuration. In the preferred embodiment, the amino acids arein the (S) or L-configuration. If non-naturally occurring side chainsare used, non-amino acid substituents may be used, for example toprevent or retard in vivo degradations.

Also encompassed by “heme oxygenase-I”, “HO-I”, etc. are homologpolypeptides having at least about 80% sequence identity, usually atleast about 85% sequence identity, preferably at least about 90%sequence identity, more preferably at least about 95% a sequenceidentity and most preferably at least about 98% sequence identity withthe polypeptide encoded by nucleotides 81-944 of the nucleotide sequenceshown in FIG. 3 (SEQ ID NO:1) and which exhibit at least one biologicalactivity that is normally associated with the human heme oxygenase-Ienzyme.

By “nucleic acid molecules that encode NO-I”, “nucleic acid moleculesencoding a polypeptide having heme oxygenase-t activity” and grammaticalequivalents thereof is meant the nucleotide sequence of human hemeoxygenase-I as shown nucleotides 81-944 of FIG. 3 (SEQ ID NO:1) as wellas nucleotide sequences having at least about 80% sequence identity,usually at least about 85% sequence identity, preferably at least about90% sequence identity, more preferably at least about 95% sequenceidentity and most preferably at least about 98% sequence identity withnucleotides 81-944 of the nucleotide sequence shown in FIG. 3 (SEQ IDNO:1) and which encode a polypeptide that exhibits at least onebiological activity that is normally associated with the human hemeoxygenase-I enzyme.

As is known in the art, a number of different programs can be used toidentify whether a protein or nucleic acid has sequence identity orsimilarity to a known sequence. Sequence identity and/or similarity isdetermined using standard techniques known in the art, including, butnot limited to, the local sequence identity algorithm of Smith &Waterman, Adv. Appl. Math. 2:482 (1981), by the sequence identityalignment algorithm of Needleman & Wunsch, J. Mol. Biol. 48:443 (1970),by the search for similarity method of Pearson & Lipman, PNAS USA85:2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Drive, Madison, Wis.), theBest Fit sequence program described by Devereux et al., Nucl. Acid Res.12:387-395 (1984), preferably using the default settings, or byinspection. Preferably, percent identity is calculated by FastDB basedupon the following parameters: mismatch penalty of 1; gap penalty of 1;gap size penalty of 0.33; and joining penalty of 30, “Current Methods inSequence Comparison and Analysis,” Macromolecule Sequencing andSynthesis, Selected Methods and Applications, pp 127-149 (1988), Alan R.Liss, Inc.

An example of a useful algorithm is PILEUP. PILEUP creates a multiplesequence alignment from a group of related sequences using progressive,pairwise alignments. It can also plot a tree showing the clusteringrelationships used to create the alignment. PILEUP uses a simplificationof the progressive alignment method of Feng & Doolittle, J. Mol. Evol.35:351-360 (1987); the method is similar to that described by Higgins &Sharp CABIOS 5:151-153 (1989). Useful PILEUP parameters including adefault gap weight of 3.00, a default gap length weight of 0.10, andweighted end gaps.

Another example of a useful algorithm is the BLAST algorithm, describedin Altschul et al., J. Mol. Biol. 215, 403-410, (1990) and Karlin etal., PNAS USA 90:5873-5787 (1993). A particularly useful BLAST programis the WU-BLAST-2 program which was obtained from Altschul et al.,Methods in Enzymology, 266: 460-480 (1996);http:/fblast.wustl/edu/blast/README.html]. WU-BLAST-2 uses severalsearch parameters, most of which are set to the default values. Theadjustable parameters are set with the following values: overlap span=1,overlap fraction=0.125, word threshold (T)=11. The HSP S and HSP S2parameters are dynamic values and arc established by the program itselfdepending upon the composition of the particular sequence andcomposition of the particular database against which the sequence ofinterest is being searched; however, the values may be adjusted toincrease sensitivity.

An additional useful algorithm is gapped BLAST as reported by Altschulet al. Nucleic Acids Res. 25:3389-3402. Gapped BLAST uses BLOSUM-62substitution scores; threshold T parameter set to 9; the two-hit methodto trigger ungapped extensions; charges gap lengths of k a cost of 10+k;X_(u) set to 16, and X_(g) set to 40 for database search stage and to 67for the output stage of the algorithms. Gapped alignments are triggeredby a score corresponding to ˜22 bits.

A % amino acid or nucleic acid sequence identity value is determined bythe number of matching identical residues divided by the total number ofresidues of the “longer” sequence in the aligned region. The “longer”sequence is the one having the most actual residues in the alignedregion (gaps introduced by WU-Blast-2 to maximize the alignment scoreare ignored).

The alignment may include the introduction of gaps in the sequences tobe aligned. In addition, for sequences which contain either more orfewer amino acids than the amino acid sequence of the polypeptideencoded by nucleotides 81-944 of the nucleotide sequence shown in FIG. 3(SEQ ID NO:1), it is understood that in one embodiment, the percentageof sequence identity will be determined based on the number of identicalamino acids in relation to the total number of amino acids. Thus, forexample, sequence identity of sequences shorter than that of thepolypeptide encoded by nucleotides 81-944 of the nucleotide sequenceshown in FIG. 3 (SEQ ID NO:1), as discussed below, will be determinedusing the number of amino acids in the shorter sequence, in oneembodiment. In percent identity calculations relative weight is notassigned to various manifestations of sequence variation, such as,insertions, deletions, substitutions, etc.

In one embodiment, only identities are scored positively (+1) and allforms of sequence variation including gaps are assigned a value of “0”,which obviates the need for a weighted scale or parameters as describedbelow for sequence similarity calculations. Percent sequence identitycan be calculated, for example, by dividing the number of matchingidentical residues by the total number of residues of the “shorter”sequence in the aligned region and multiplying by 100. The “longer”sequence is the one having the most actual residues in the alignedregion.

Heme oxygenase-I having less than 100% sequence identity with thepolypeptide encoded by nucleotides 81-944 of the nucleotide sequenceshown in FIG. 3 (SEQ ID NO:1) will generally be produced from nativeheme oxygenase-I nucleotide sequences from species other than human andvariants of native heme oxygenase-I nucleotide sequences from human ornon-human sources. In this regard, it is noted that many techniques arewell known in the art and may be routinely employed to producenucleotide sequence variants of native heme oxygenase-I sequences andassaying the polypeptide products of those variants for the presence ofat least one activity that is normally associated with a native hemeoxygenase-I polypeptide.

Polypeptides having heme oxygenase-I activity may be shorter or longerthan the polypeptide encoded by nucleotides 81-944 of the nucleotidesequence shown in FIG. 3 (SEQ ID NO:1). Thus, in a preferred embodiment,included within the definition of heme oxygenase-I polypeptide areportions or fragments of the polypeptide encoded by nucleotides 81-944of the nucleotide sequence shown in FIG. 3 (SEQ ID NO:1). In oneembodiment herein, fragments of the polypeptide encoded by nucleotides81-944 of the nucleotide sequence shown in FIG. 3 (SEQ ID NO:1) areconsidered heme oxygenase-I polypeptides if a) they have at least theindicated sequence identity; and b) preferably have a biologicalactivity of naturally occurring heme oxygenase-I, as described above.

In addition, as is more fully outlined below, heme oxygenase-I can bemade longer than the polypeptide encoded by nucleotides 81-944 of thenucleotide sequence shown in FIG. 3 (SEQ ID NO:1); for example, by theaddition of other fusion sequences, or the elucidation of additionalcoding and non-coding sequences.

The heme oxygenase-I polypeptides are preferably recombinant. A“recombinant polypeptide” is a polypeptide made using recombinanttechniques, i.e. through the expression of a recombinant nucleic acid asdescribed below. In a preferred embodiment, the heme oxygenase-I of theinvention is made through the expression of nucleotides 81-944 of thenucleotide sequence shown in FIG. 3 (SEQ ID NO:1), or fragment thereof.A recombinant polypeptide is distinguished from naturally occurringprotein by at least one or more characteristics. For example, thepolypeptide may be isolated or purified away from some or all of theproteins and compounds with which it is normally associated in its wildtype host, and thus may be substantially pure. For example, an isolatedpolypeptide is unaccompanied by at least some of the material with whichit is normally associated in its natural state, preferably constitutingat least about 0.5%, more preferably at least about 5% by weight of thetotal protein in a given sample. A substantially pure polypeptidecomprises at least about 75% by weight of the total polypeptide, with atleast about 80% being preferred, and at least about 90% beingparticularly preferred. The definition includes the production of a hemeoxygenase-I polypeptide from one organism in a different organism orhost cell. Alternatively, the polypeptide may be made at a significantlyhigher concentration than is normally seen, through the use of ainducible promoter or high expression promoter, such that thepolypeptide is made at increased concentration levels. Alternatively,the polypeptide may be in a form not normally found in nature, as in theaddition of amino acid substitutions, insertions and deletions, asdiscussed below.

As used herein and further defined below, “nucleic acid” may refer toeither DNA or RNA, or molecules which contain both deoxy- andribonucleotides. The nucleic acids include genomic DNA, cDNA andoligonucleotides including sense and anti-sense nucleic acids. Suchnucleic acids may also contain modifications in the ribose-phosphatebackbone to increase stability and half life of such molecules inphysiological environments.

The nucleic acid may be double stranded, single stranded, or containportions of both double stranded or single stranded sequence. As will beappreciated by those in the art, the depiction of a single strand(“Watson”) also defines the sequence of the other strand (“Crick”); thusthe sequences depicted in FIGS. 1 and 3 also include the complement ofthe sequence. By the term “recombinant nucleic acid” herein is meantnucleic acid, originally formed in vitro, in general, by themanipulation of nucleic acid by endonucleases, in a form not normallyfound in nature. Thus an isolated nucleic acid, in a linear form, or anexpression vector formed in vitro by ligating DNA molecules that are notnormally joined, are both considered recombinant for the purposes ofthis invention. It is understood that once a recombinant nucleic acid ismade and reintroduced into a host cell or organism, it will replicatenon-recombinantly, i.e. using the in vivo cellular machinery of the hostcell rather than in vitro manipulations; however, such nucleic acids,once produced recombinantly, although subsequently replicatednon-recombinantly, are still considered recombinant for the purposes ofthe invention.

In one embodiment, the present invention provides nucleic acids encodingheme oxygenase-I variants. These variants fall into one or more of threeclasses: substitutional, insertional or deletional variants. Thesevariants ordinarily are prepared by site specific mutagenesis ofnucleotides in nucleotides 81-944 of the nucleic acid shown in FIG. 3(SEQ ID NO:1), using cassette or PCR mutagenesis or other techniqueswell known in the art, to produce DNA encoding the variant, andthereafter expressing the DNA in a transplant graft, as described below,or a recombinant cell culture as outlined above. Amino acid sequencevariants are characterized by the predetermined nature of the variation,a feature that sets them apart from naturally occurring allelic orinterspecies variation of the heme oxygenase-I amino acid sequence. Thevariants typically exhibit the same qualitative biological activity asthe naturally occurring analogue, although variants can also be selectedwhich have modified characteristics as will be more fully outlinedbelow.

While the site or region for introducing a sequence variation ispredetermined, the mutation per se need not be predetermined. Forexample, in order to optimize the performance of a mutation at a givensite, random mutagenesis may be conducted at the target codon or regionand the expressed variants screened for the optimal desired activity.Techniques for making substitution mutations at predetermined sites inDNA having a known sequence are well known, for example, M13 primermutagenesis and PCR mutagenesis. Another example of a technique formaking variants is the method of gene shuffling, whereby fragments ofsimilar variants of a nucleotide sequence are allowed to recombine toproduce new variant combinations. Examples of such techniques are foundin U.S. Pat. Nos. 5,605,703; 5,811,238; 5,873,458; 5,830,696; 5,939,250;5,763,239; 5,965,408; and 5,945,325, each of which is incorporated byreference herein in its entirety. Screening of the mutants is done usingassays of heme oxygenase activities, as described above.

Amino acid substitutions are typically of single residues; insertionsusually will be on the order of from about 1 to 20 amino acids, althoughconsiderably larger insertions may be tolerated. Deletions range fromabout 1 to about 20 residues, although in some cases deletions may bemuch larger.

Substitutions, deletions, insertions or any combination thereof may beused to arrive at a final derivative. Generally these changes are doneon a few amino acids to minimize the alteration of the molecule.However, larger changes may be tolerated in certain circumstances. Whensmall alterations in the characteristics of the heme oxygenase-I aredesired, substitutions are generally made in accordance with thefollowing chart:

CHART I Original Residue Exemplary Substitutions Ala Ser Arg Lys AsnGln, His Asp Glu Cys Ser Gln Asn Glu Asp Gly Pro His Asn, Gln Ile Leu,Val Leu Ile, Val Lys Arg, Gln, Glu Met Leu, Ile Phe Met, Leu, Tyr SerThr Thr Ser Trp Tyr Tyr Trp, Phe Val Ile, Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those shown inChart I. For example, substitutions may be made which more significantlyaffect: the structure of the polypeptide backbone in the area of thealteration, for example the alpha-helical or beta-sheet structure; thecharge or hydrophobicity of the molecule at the target site; or the bulkof the side chain. The substitutions which in general are expected toproduce the greatest changes in the polypeptide's properties are thosein which (a) a hydrophilic residue, e.g. seryl or threonyl, issubstituted for (or by) a hydrophobic residue, e.g. leucyl, isoleucyl,phenylalanyl, valyl or alanyl; (b) a cysteine or proline is substitutedfor (or by) any other residue; (c) a residue having an electropositiveside chain, e.g. lysyl, arginyl, or histidyl, is substituted for (or by)an electronegative residue, e.g. glutamyl or aspartyl; or (d) a residuehaving a bulky side chain, e.g. phenylalanine, is substituted for (orby) one not having a side chain, e.g. glycine.

The variants typically exhibit the same qualitative biological activityand will elicit the same immune response as the naturally-occurringanalogue, although variants also are selected to modify thecharacteristics of the heme oxygenase-I as needed.

Alternatively, the variant may be designed such that the biologicalactivity of the protein is altered.

One type of covalent modification of a polypeptide included within thescope of this invention comprises altering the native glycosylationpattern of the polypeptide. “Altering the native glycosylation pattern”is intended for purposes herein to mean deleting one or morecarbohydrate moieties found in native sequence heme oxygenase-Ipolypeptide, and/or adding one or more glycosylation sites that are notpresent in the native sequence polypeptide.

Addition of glycosylation sites to polypeptides may be accomplished byaltering the amino acid sequence thereof. The alteration may be made,for example, by the addition of, or substitution by, one or more serineor threonine residues to the native sequence polypeptide (for O-linkedglycosylation sites). The amino acid sequence may optionally be alteredthrough changes at the DNA level, particularly by mutating the DNAencoding the polypeptide at preselected bases such that codons aregenerated that will translate into the desired amino acids.

Removal of carbohydrate moieties present on the polypeptide may beaccomplished by mutational substitution of codons encoding for aminoacid residues that serve as targets for glycosylation.

To produce HO-I protein to test for heme oxygenase activity, hemeoxygenase-I is cloned and expressed as outlined below. Thus, probe ordegenerate polymerase chain reaction (PCR) primer sequences may be usedto find other related heme oxygenase-I polypeptides from humans or otherorganisms. As will be appreciated by those in the art, particularlyuseful probe and/or PCR primer sequences include the unique areas of thenucleic acid sequence shown in FIG. 3 (SEQ ID NO:1). As is generallyknown in the art, preferred PCR primers are from about 15 to about 35nucleotides in length, with from about 20 to about 30 being preferred,and may contain inosine as needed. The conditions for the PCR reactionare well known in the art. It is therefore also understood that providedalong with the sequences provided herein are portions of thosesequences, wherein unique portions of 15 nucleotides or more areparticularly preferred. The skilled artisan can routinely synthesize orcut a nucleotide sequence to the desired length.

Once isolated from its natural source, e.g., contained within a plasmidor other vector or excised therefrom as a linear nucleic acid segment,the recombinant nucleic acid can be further-used as a probe to identifyand isolate other nucleic acids. It can also be used as a “precursor”nucleic acid to make modified or variant nucleic acids and proteins.

Using the nucleic acids of the present invention which encode a protein,a variety of expression vectors can be made. The expression vectors maybe either self-replicating extrachromosomal vectors or vectors whichintegrate into a host genome. Generally, these expression vectorsinclude transcriptional and translational regulatory nucleic acidoperably linked to the nucleic acid encoding the protein. The term“control sequences” refers to DNA sequences necessary for the expressionof an operably linked coding sequence in a particular host organism. Thecontrol sequences that are suitable for prokaryotes, for example,include a promoter, optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Nucleic acid is “operably linked” when it is placed into a functionalrelationship with another nucleic acid sequence. For example, DNA for apresequence or secretory leader is operably linked to DNA for apolypeptide if it is expressed as a preprotein that participates in thesecretion of the polypeptide; a promoter or enhancer is operably linkedto a coding sequence if it affects the transcription of the sequence; ora ribosome binding site is operably linked to a coding sequence if it ispositioned so as to facilitate translation. As another example, operablylinked refers to DNA sequences linked so as to be contiguous, and, inthe case of a secretory leader, contiguous and in reading phase.However, enhancers do not have to be contiguous. Linking is accomplishedby ligation at convenient restriction sites. If such sites do not exist,the synthetic oligonucleotide adaptors or linkers are used in accordancewith conventional practice. The transcriptional and translationalregulatory nucleic acid will generally be appropriate to the host cellused to express the heme oxygenase-I; for example, transcriptional andtranslational regulatory nucleic acid sequences from Bacillus arepreferably used to express the heme oxygenase-I in Bacillus. Numeroustypes of appropriate expression vectors, and suitable regulatorysequences are known in the art for a variety of host cells.

In general, the transcriptional and translational regulatory sequencesmay include, but are not limited to, promoter sequences, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, and enhancer or activator sequences. In apreferred embodiment, the regulatory sequences include a promoter andtranscriptional start and stop sequences.

Promoter sequences encode either constitutive or inducible promoters.The promoters may be either naturally occurring promoters or hybridpromoters. Hybrid promoters, which combine elements of more than onepromoter, are also known in the art, and are useful in the presentinvention.

In addition, the expression vector may comprise additional elements. Forexample, the expression vector may have two replication systems, thusallowing it to be maintained in two organisms, for example in mammalianor insect cells for expression and in a procaryotic host for cloning andamplification. Furthermore, for integrating expression vectors, theexpression vector contains at least one sequence homologous to the hostcell genome, and preferably two homologous sequences which flank theexpression construct. The integrating vector may be directed to aspecific locus in the host cell by selecting the appropriate homologoussequence for inclusion in the vector. Constructs for integrating vectorsare well known in the art.

In addition, in a preferred embodiment, the expression vector contains aselectable marker gene to allow the selection of transformed host cells.Selection genes are well known in the art and will vary with the hostcell used.

A preferred expression vector system is a retroviral vector system suchas is generally described in PCT/US97/01019 and PCT/US97/01048, both ofwhich are hereby expressly incorporated by reference.

Proteins of the present invention are produced by culturing a host celltransformed with an expression vector containing nucleic acid encodingthe protein, under the appropriate conditions to induce or causeexpression of the protein. The conditions appropriate for proteinexpression will vary with the choice of the expression vector and thehost cell, and will be easily ascertained by one skilled in the artthrough routine experimentation. For example, the use of constitutivepromoters in the expression vector may require optimizing the growth andproliferation of the host cell, while the use of an inducible promoterrequires the appropriate conditions for induction. In addition, in someembodiments, the timing of the harvest is important. For example, thebaculoviral systems used in insect cell expression are lytic viruses,and thus harvest time selection can be crucial for product yield.

Appropriate host cells include yeast, bacteria, archaebacteria, fungi,and insect and animal cells, including mammalian cells. Of particularinterest are Drosophila melanogaster cells, Saccharomyces cerevisiae andother yeasts, E. coli, Bacillus subtilis, SF9 cells, C129 cells, 293cells, Neurospora, BHK, CHO, COS, and HeLa cells, fibroblasts, Schwanomacell lines, immortalized mammalian myeloid and lymphoid cell lines,tumor cell lines, and B lymphocytes.

In a preferred embodiment, the proteins are expressed in mammaliancells. Mammalian expression systems are also known in the art, andinclude retroviral systems. A mammalian promoter is any DNA sequencecapable of binding mammalian RNA polymerase and initiating thedownstream (3′) transcription of a coding sequence for a protein intomRNA. A promoter will have a transcription initiating region, which isusually placed proximal to the 5′ end of the coding sequence, and a TATAbox, using a located 25-30 base pairs upstream of the transcriptioninitiation site. The TATA box is thought to direct RNA polymerase II tobegin RNA synthesis at the correct site. A mammalian promoter will alsocontain an upstream promoter element (enhancer element), typicallylocated within 100 to 200 base pairs upstream of the TATA box. Anupstream promoter element determines the rate at which transcription isinitiated and can act in either orientation. Of particular use asmammalian promoters are the promoters from mammalian viral genes, sincethe viral genes are often highly expressed and have a broad host range.Examples include the SV40 early promoter, mouse mammary tumor virus LTRpromoter, adenovirus major late promoter, herpes simplex virus promoter,and the CMV promoter.

Typically, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-translational cleavage and polyadenylation.Examples of transcription terminator and polyadenlytion signals includethose derived form SV40.

The methods of introducing exogenous nucleic acid into mammalian hosts,as well as other hosts, is well known in the art, and will vary with thehost cell used. Techniques include dextran-mediated transfection,calcium phosphate precipitation, polybrene mediated transfection,protoplast fusion, electroporation, viral infection, encapsulation ofthe polynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

Proteins may be expressed in bacterial systems. Bacterial expressionsystems are well known in the art.

A suitable bacterial promoter is any nucleic acid sequence capable ofbinding bacterial RNA polymerase and initiating the downstream (3′)transcription of the coding sequence of cell cycle protein into mRNA. Abacterial promoter has a transcription initiation region which isusually placed proximal to the 5′ end of the coding sequence. Thistranscription initiation region typically includes an RNA polymerasebinding site and a transcription initiation site. Sequences encodingmetabolic pathway enzymes provide particularly useful promotersequences. Examples include promoter sequences derived from sugarmetabolizing enzymes, such as galactose, lactose and maltose, andsequences derived from biosynthetic enzymes such as tryptophan.Promoters from bacteriophage may also be used and are known in the art.In addition, synthetic promoters and hybrid promoters are also useful;for example, the tac promoter is a hybrid of the trp and lac promotersequences. Furthermore, a bacterial promoter can include naturallyoccurring promoters of non-bacterial origin that have the ability tobind bacterial RNA polymerase and initiate transcription.

In addition to a functioning promoter sequence, an efficient ribosomebinding site is desirable. In E. coli, the ribosome binding site iscalled the Shine-Delgarno (SD) sequence and includes an initiation codonand a sequence 3-9 nucleotides in length located 3-11 nucleotidesupstream of the initiation codon.

The expression vector may also include a signal peptide sequence thatprovides for secretion of the protein in bacteria. The signal sequencetypically encodes a signal peptide comprised of hydrophobic amino acidswhich direct the secretion of the protein from the cell, as is wellknown in the art. The protein is either secreted into the growth media(gram-positive bacteria) or into the periplasmic space, located betweenthe inner and outer membrane of the cell (gram-negative bacteria).

The bacterial expression vector may also include a selectable markergene to allow for the selection of bacterial strains that have beentransformed. Suitable selection genes include genes which render thebacteria resistant to drugs such as ampicillin, chloramphenicol,erythromycin, kanamycin, neomycin and tetracycline. Selectable markersalso include biosynthetic genes, such as those in the histidine,tryptophan and leucine biosynthetic pathways.

These components are assembled into expression vectors. Expressionvectors for bacteria are well known in the art, and include vectors forBacillus subtilis, E. coli, Streptococcus cremoris, and Streptococcuslividans, among others.

The bacterial expression vectors are transformed into bacterialhostcells using techniques well known in the art, such as calciumchloride treatment, electroporation, and others.

Proteins may produced in insect cells. Expression vectors for thetransformation of insect cells, and in particular, baculovirus-basedexpression vectors, are well known in the art.

Proteins may also produced in yeast cells. Yeast expression systems arewell known in the art, and include expression vectors for Saccharomycescerevisiae, Candida albicans and C. maltosa, Hansenula polymorpha,Kluyveromyces fragilis and K. lactis, Pichia guillerimondii and P.pastoris, Schizosaccharomyces pombe, and Yarrowia lipolytica. Preferredpromoter sequences for expression in yeast include the inducible GAL1,10promoter, the promoters from alcohol dehydrogenase, enolase,glucokinase, glucose-6-phosphate isomerase,glyceraldehyde-3-phosphate-dehydrogenase, hexokinase,phosphofructokinase, 3-phosphoglycerate mutase, pyruvate kinase, and theacid phosphatase gene. Yeast selectable markers include ADE2, HIS4,LEU2, TRP1, and ALG7, which confers resistance to tunicamycin; theneomycin phosphotransferase gene, which confers resistance to G418; andthe CUP1 gene, which allows yeast to grow in the presence of copperions.

The protein may also be made as a fusion protein, using techniques wellknown in the art. Thus, for example, the protein may be made as a fusionprotein to increase expression, or for other reasons. For example, whenthe protein is a peptide, the nucleic acid encoding the peptide may belinked to other nucleic acid for expression purposes.

To test for heme oxygenase activity, the protein is purified or isolatedafter expression. Proteins may be isolated or purified in a variety ofways known to those skilled in the art depending on what othercomponents are present in the sample. Standard purification methodsinclude electrophoretic, molecular, immunological and chromatographictechniques, including ion exchange, hydrophobic, affinity, andreverse-phase HPLC chromatography, and chromatofocusing. For example,the heme oxygenase protein may be purified using a standard anti-hemeoxygenase antibody column. Ultrafiltration and diafiltration techniques,in conjunction with protein concentration, are also useful. For generalguidance in suitable purification techniques, see Scopes, R., ProteinPurification, Springer-Verlag, N.Y. (1982). The degree of purificationnecessary will vary depending on the use of the heme oxygenase-Iprotein. In some instances no purification will be necessary.

Nucleic acid molecules encoding heme oxygenase-I as well as any nucleicacid molecule derived from either the coding or non-coding strand of anucleic acid molecule that encodes heme oxygenase-I may be contactedwith cells of a organ transplant in a variety of ways which are knownand routinely employed in the art, wherein the contacting may be ex vivoor in vivo. The particular protocol will depend upon the nature of theorgan, the form of the nucleic acid, and the use of immunosuppressantsor other drugs.

By the term “conditions permissive for the contacting of exogenousnucleic acid”, and grammatical equivalents herein is meant conditionswhich allow cells of the ex vivo or in vivo organ transplant to becontacted with the exogenous nucleic acid, whereby heme oxygenaseactivity is modified. In a preferred embodiment, contacting results inthe uptake of the nucleic acid into the cells.

In a preferred embodiment, the nucleic acid encodes a protein which isexpressed. In some embodiments, the expression of the exogeneous nucleicacid is transient; that is, the exogeneous protein is expressed for alimited time. In other embodiments, the expression is permanent

In some embodiments, the exogeneous nucleic acid is incorporated intothe genome of the target cell; for example, retroviral vectors describedbelow integrate into the genome of the host cell. Generally this is donewhen longer or permanent expression is desired. In other embodiments,the exogeneous nucleic acid does not incorporate into the genome of thetarget cell but rather exists autonomously in the cell; for example,many such plasmids are known. This embodiment may be preferable whentransient expression is desired.

The permissive conditions will depend on the form of the exogenousnucleic acid. The production of various expression vectors is describedabove. Thus, for example, when the exogenous nucleic acid is in the formof an adenoviral, retroviral, or adenoassociated viral vector, thepermissive conditions are those which allow viral contact and/orinfection of the cell. Similarly, when the exogenous nucleic acid is inthe form of a plasmid, the permissive conditions allow the plasmid tocontact or enter the cell. Thus, the form of the exogenous nucleic acidand the conditions which are permissive for contacting are correlated.These conditions are generally well known in the art.

Permissive conditions depend on the expression vector to be used, theamount of expression desired and the target cell. Generally, conditionswhich allow in vitro uptake of exogenous cells work for ex vivo and invivo cells.

Permissive conditions are analyzed using well-known techniques in theart. For example, the expression of exogenous nucleic acid may beassayed by detecting the presence of mRNA, using Northern hybridization,or protein, using antibodies or biological function assays.

Specific conditions for the uptake of exogenous nucleic acid are wellknown in the art. They include, but are not limited to, retroviralinfection, adenoviral infection, transformation with plasmids,transformation with liposomes containing exogenous nucleic acid,biolistic nucleic acid delivery (i.e. loading the nucleic acid onto goldor other metal particles and shooting or injecting into the cells),adenoassociated virus infection, HIV virus infection and Epstein-Barrvirus infection. These may all be considered “expression vectors” forthe purposes of the invention.

The expression vectors may be either extrachromosomal vectors or vectorswhich integrate into a host genome as outlined above. Generally, theseexpression vectors include transcriptional and translational regulatorynucleic acid operably linked to the exogenous nucleic acid. “Operablylinked” in this context means that the transcriptional and translationalregulatory DNA is positioned relative to the coding sequence of theexogenous protein in such a manner that transcription is initiated.Generally, this will mean that the promoter and transcriptionalinitiation or start sequences are positioned 5′ to the exogenous proteincoding region. The transcriptional and translational regulatory nucleicacid will generally be appropriate to the host cell in which theexogenous protein is expressed; for example, transcriptional andtranslational regulatory nucleic acid sequences from mammalian cells,and particularly humans, are preferably used to express the exogenousprotein in mammals and humans. Numerous types of appropriate expressionvectors, and suitable regulatory sequences are known in the art.

In general, the transcriptional and translational regulatory sequencesmay include, but are not limited to, promoter sequences, ribosomalbinding sites, transcriptional start and stop sequences, translationalstart and stop sequences, and enhancer or activator sequences. In apreferred embodiment, the regulatory sequences include a promoter andtranscriptional start and stop sequences.

Promoter sequences encode either constitutive, tissue specific orinducible promoters. The promoters may be either naturally occurringpromoters or hybrid promoters. Hybrid promoters, which combine elementsof more than one promoter, are also known in the art, and are useful inthe present invention.

In addition, the expression vector may comprise additional elements. Forexample, for integrating expression vectors, the expression vectorcontains at least one sequence homologous to the host cell genome, andpreferably two homologous sequences which flank the expressionconstruct. The integrating vector may be directed to a specific locus inthe host cell by selecting the appropriate homologous sequence forinclusion in the vector. Constructs for integrating vectors are wellknown in the art.

Suitable retroviral vectors include LNL6, LXSN, and LNCX (see Byun etal., Gene Ther. 3(9):780-8 (1996 for review).

In a preferred embodiment, the nucleic acids are contacted cells of atransplant organ in the form of an adenovirus. Suitable adenoviralvectors include modifications of human adenoviruses such as Ad2 or Ad5,wherein genetic elements necessary for the virus to replicate in vivohave been removed; e.g. the E1 region, and an expression cassette codingfor the exogenous gene of interest inserted into the adenoviral genome(for example Ad_(GV)CFTR₁₀).

In one embodiment of the present invention, the nucleic acid molecule isintroduced into cells of the organ transplant by liposome-mediatednucleic acid transfer. In this regard, many liposome-based reagents arewell known in the art, are commercially available and may be routinelyemployed for introducing a nucleic acid molecule into cells of the organtransplant. Certain embodiments of the present invention will employcationic lipid transfer vehicles such as Lipofectamine or Lipofectin(Life Technologies), dioleoylphosphatidylethanolamine (DOPE) togetherwith a cationic cholesterol derivative (DC cholesterol),N[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)(Sioud et al., J. Mol. Biol. 242:831-835 (1991)), DOSPA:DOPE, DOTAP,DMRIE:cholesterol, DDAB:DOPE, and the like. Production of liposomeencapsulated nucleic acid is well known in the art and typicallyinvolves the combination of lipid and nucleic acid in a ratio of about1:1.

In vivo delivery includes, but is not limited to direct injection intothe organ, via catheter, or by other means of perfusion. The nucleicacid may be administered intravascularly at a proximal location to thetransplant organ or administered systemically. One of ordinary skill inthe art will recognized the advantages and disadvantages of each mode ofdelivery. For instance, direct injection may produce the greatest titerof nucleic acid in the organ, but distribution of the nucleic acid willlikely be uneven throughout the organ. Introduction of the nucleic acidproximal to the transplant organ will generally result in greatercontact with the cells of the organ, but systemic administration isgenerally much simpler. Administration may also be to the donor prior toremoval of the organ. The nucleic acids may be introduced in a singleadministration, or several administrations, beginning before removal ofthe organ from the donor as well as after transplantation. The skilledartisan will be able to determine a satisfactory means of delivery anddelivery regimen without undue experimentation.

Nucleic acids may be contacted with cells of the transplant organ exvivo. When bathing the organ in a composition comprising the nucleicacids, conventional medium may be sued, such as organ preservationsolution. The temperature at which the the organ may be maintained willbe conventional, typically in the range of about 1° to 8° C. Theresidence time of the organ in the medium will generally be in the rangeof about 10 minutes to 48 hours, more usually about 10 minutes to 2hours. The nucleic acids may be contacted with cells of the organ invivo as well as ex vivo.

In a preferred embodiment, the nucleic acid is contacted with cells of aorgan transplant by direct injection into the transplanted organ. Inthis regard, it is well known in the art that living cells are capableof internalizing and incorporating exogenous nucleic acid molecule withwhich the cells come in contact. That nucleic acid may then be expressedby the cell that has incorporated it into its nucleus.

In a preferred embodiment, the nucleic acid is contacted with cells of atransplant organ by intravascular injection proximate to the transplantorgan. In an alternate preferred embodiment, the nucleic acid iscontacted with cells of a transplant organ by systemic administration.

The above described nucleic acid molecules will function to modulate theoverall heme oxygenase-I activity of a cell with which it is contacted.In cases where the nucleic acid molecule encodes a polypeptide having atleast one activity normally associated with the human heme oxygenase-Ipolypeptide, the modulation will generally be exemplified by an increasein the heme oxygenase-I activity of the cell in which the nucleic acidmolecule is expressed. In cases where the nucleic acid molecule is anantisensc heme oxygenase-I oligonucleotide, the modulation willgenerally be exemplified by a decrease in the heme oxygenase-I activityof the cell into which the nucleic acid molecule is introduced.

The subject nucleic acids may be used with a wide variety of hosts,particularly primates, more particularly humans, or with domesticanimals. The subject nucleic acids may be used in conjunction with thetransplantation of a wide variety of organs, including, but not limitedto, kidney, heart, liver, spleen, bone marrow, pancreas, lung, and isletof langerhans. The subject nucleic acids may be used for allogenic, aswell as xenogenic, grafts.

The subject nucleic acids may be used as adjunctive therapy withimmunosuppressant compounds, such as cyclosporin, FK506, MHC class Ioligopeptides, or other immunosuppressants. Such adjunct use may allowreduced amounts of the immunosuppressant to be used than would be usedotherwise.

Generally, the graft life will be extended for a significant amount oftime beyond what could normally be anticipated in the absence of thesubject nucleic acids, more usually at least five days. The actualamount of time transplant life is extended will vary with the variousconditions of the procedure, particularly depending on the organ type tobe transplanted. This can be useful in areas where xenogeneic graftshave been used awaiting an allogenic graft, to allow for reduced amountsof immunosuppressants or avoid using immunosuppressants altogether.

EXPERIMENTAL

The following examples are offered by illustration and not by way oflimitation.

Example 1 Metalloprotoporphyrin-Induced Immunomodulation

Materials and Methods

Animals: Male, 7-8 week old CBA/J (H-2^(l)) and C57BL/6/J (H-2^(b)) micewere purchased from the Jackson Laboratory (Bar Harbor, Me.). Mice weremaintained in our animal facility following Animal Welfare Guideline,Department of Health, CA.

Synthetic Metalloporphyrins: Various synthetic metalloporphyrins werepurchased from Porphyrin Products, Inc. (Logan, Utah). They weredissolved in 0.2 M NaOH, adjusted to pH 74 with 1 M HCl and subsequentlydiluted to 1 mg/ml in PBS.

Cytoioxic T-cell Activity: To assay the effect of metalloprotoporphyrinson cytotoxic T-cell activity, CBA to B6 effectors were generatedfollowing a five day culture of 3×10⁶ CBA spleen cells with 3×10⁶mitomycin-treated B6 spleen cells in wells of a 24-well plate (Nunclone,Delta, Nunc, Denmark) in R-10 medium. Effector cells were then harvestedand washed. (H2b), a mouse lymphoma induced in C57BL/6N was used astarget cells. EL4 cells were routinely subcultured once every threedays. They were then collected, washed, and labeled with 0.1 mCi ofsodium chromate-51 in 200 μl for one hour at 37° C. Effector (E) andtarget (t) cells were added into V-shaped tissue culture plates (Nunc,Denmark) at E:T of 20:1. Metalloprotoporphyrins were diluted to theworking concentrations with PBS and added at the beginning of the fourhour incubation period. For the determination of maximal release, 1%triton X-100 was added to separate wells. Plates were centrifuged forthree minutes to increase cellular contact before the four hourincubation period. After incubation 75 μl supernatant from each well wascollected and the amount of ⁵¹Cr released was counted using a TopCountscintillation counter. The degree of cell lysis was calculated using theformula below:${\%\quad{lysis}} - {\frac{{CPM}_{experimental} - {CPM}_{spontaneous}}{{CPM}_{total} - {CPM}_{spontaneous}} \times 100}$

Heterotopic Heart Transplantation: Abdominal heterotopic hearttransplantation was performed as previously described by Ono and Lindsey(J. Thorac. Cardiovasc. Surg. 1969 7:225-229) using C56Bl/6 donors andCBA recipients. Metalloporphyrin was administered intraperitoneallyusing various protocols. Heart allograft survival was monitored daily bydirect palpation, and rejection was defined as termination of palpablecardiac contractility. Results are expressed as percentage graftsurvival at a given postoperative period. Statistical analysis wasperformed with the Mann-Whitney test.

Results

Zinc- and cobalt-protoporphyrin inhibit cytotoxicity in vitro. Theeffect of Zn- and Co-protoporphyrins on T- and NK-cell mediatedcytotoxicity was evaluated in an in vitro four hour chromium releaseassay. Results of a representative experiment using cytotoxic T-cellsare shown in FIG. 1. Similar results were observed in NK-cell assays.Addition, of protoporphyrin to the tissue culture inhibited target celllysis in a dose dependent manner. At about 10 μg/ml target cell lysiswas inhibited completely (0% lysis). At even higher concentrations,chromium release from target cells in the presence of protoporphyrinswas lower than the spontaneous release observed in the absence of thecompounds. These results demonstrate modulation of HO activity bymetalloporphyrins results in inhibition of cytotoxicity in vitro.

Zinc- and cobalt-protoporphyrin therapy of heart allograft recipientsresults in prolongation of graft survival. The effect ofmetalloprotoporphyrins therapy on heart allograft survival was evaluatedin a mouse model. CBA recipients of C57Bl/6 hearts were treatedfollowing transplantation with several doses of Zn- orco-protoporphyrin. Compared to control animals (mean survivaltime=7.8±1.1) heart allograft survival was significantly prolonged to12.0±2.4 (p=0.008) and 10.5±0.6 (p=0.004) days in Zn- orCo-protoporphyrin treated animals, respectively. Pre-treatment of heartdonors one day before transplantation resulted in a prolongation ofgraft survival to 10.3±1.5 days (p=0.03).

It is evident from the above results, that by usingmetalloprotoporphyrins, one can greatly extend the survival of implantsin a host. The compounds have few side effects and can be used'safelywith positive results.

Example 2 Heme Oxvgenase-I Nucleic Acid-Induced Immunomodulation

In Example 1 it is demonstrated that upregulation of heme oxygenase indonor hearts results in prolongation of heart allograft survival. Toevaluate if transfection of donor hearts with a cDNA encoding hemeoxygcnase-I results in elevated heme oxygenase-I activity and prolongedheartallograft survival, a cDNA encoding the human heme oxygenase-Ipolypeptide (for the nucleic acid sequence, see nucleotides 81-944 ofFIG. 3 (SEQ ID NO:1)) is cloned into a plasmid expression vector,wherein expression of the gene is under transcriptional control of theCMV promoter. Plasmid DNA is then mixed with Lipofectin reagent anddiluted in a 5% glucose solution. The hearts are then excised from LEWrecipients and flushed with 5% glucose solution, followed by thesolution containing the DNA/liposome particles. After incubation for 10minutes, the transfected hearts are transplanted heterotopically intoACI recipients. Allograft survival is monitored daily by directpalpation and compared with untreated grafts.

Example 3 Extended Survival of Orthotonic Liver Transplants withAdenovirus HO-I Transfected Livers

Materials and Methods

Animals. Genetically obese (fa/fa) male Zucker rats (220-275 g) and lean(fa/−) Zucker rats (250-300 g) were used (Harlan Sprague Dawley Inc.,Indianapolis, Ind., USA). Animals were fed standard rodent chow andwater ad libitum and cared for according to guidelines approved by theAmerican Association of Laboratory Animal Care.

Synthetic metalloporphyrins. Metalloporphyrins (CoPP and ZnPP) werepurchased from Porphyrin Products Inc. (Logan, Utah, USA). They weredissolved in 0.2 M NaOH, subsequently adjusted to a pH of 7.4, anddiluted in 0.85% NaCl. The stock concentration of metalloporphyrins was1 mg/mL.

Ad-HO-I construct. A 1.0-kbp Xhol-HindIII fragment from the rat HO-IcDNA clone pRHO-I, containing the entire coding region was cloned intoplasmid pAC-CMVpLpA. Ad-HO-I was generated by homologous recombinationin 911 cells after cotransfection with the pAC-HO-I plasmid and plasmidpJM17. The recombinant Ad-HO-I clones were screened by Southern blotanalysis. Ad containing the Escherichia coli β-galactosidase gene (Ad-βGal) is well known in the art.

Syngeneic Orthotopic Liver Transplant (OLT) model. Syngeneic livertransplants were performed using fatty livers that were harvested fromobese Zucker rats and stored for 4 hours at 4° C. in University ofWisconsin (UW) solution before being transplanted into lean Zuckerrecipients. OLTs were performed with revascularization without hepaticartery reconstruction. There were 2 treatment groups. In the firstgroup, obese

Zucker rats (n=10) received CoPP (5 mg/kg intraperitoneally) 24 hoursbefore the procurement. Group 2 donors (n=11) were treated with Ad-HO-I(2.4×10⁹ pfu intravenously) 24 hours before harvest. OLT recipients werefollowed for survival and serum glutamic-oxaloacetic trans-aminase(sGOT) levels. Separate groups of rats (n=2/group) were sacrificed at 1,7, 14, and 100 days after OLT, and liver samples were collected forH&E/immunohistology staining and Western blot analysis.

Histology and immunohistochemistry. Liver specimens were fixed in a 10%buffered formalin solution and embedded in paraffin. Sections were madeat 4 μm and stained with H&E. The previously published Suzuki's criteria(Suzuki et al., Transplantation 55:1265-1272 (1993)), which usesneutrophil infiltration as a measure of liver injury, were modified toevaluate the histologic severity of I/R injury in the OLT model. In thisclassification sinusoidal congestion, hepatocyte necrosis, andballooning degeneration are graded from 0 to 4. No necrosis, congestionor centrilobular ballooning is given a score of 0, whereas severecongestion and ballooning degeneration as well as greater than 60%lobular necrosis is given a value of 4.

OLTs were examined serially by immunohistochemistry for mononuclear cell(MNC) infiltration. Briefly, liver tissue was embedded in Tissue Tek OCTcompound (Miles Inc., Elkhart, Ind., USA), snap frozen in liquidnitrogen, and stored at −70° C. Cryostat sections (5 μm) were fixed inacetone, and then endogenous peroxidase activity was inhibited with 0.3%H₂O₂ in PBS. Normal heat-inactivated donkey serum (10%) was used forblocking. Appropriate primary mouse Ab against rat T cells (R73) andmonocytes/macrophages (ED1) (Harlan Bioproducts for Science,Indianapolis, Ind., USA) were added at optimal dilutions. Bound primaryAb was detected using biotinylated donkey anti-mouse IgG andstreptavidin peroxidase—conjugated complexes (DAKO Corp., Carpinteria,Calif., USA). The control sections were performed by replacing theprimary Ab with either dilution buffer or normal mouse serum. Theperoxidase reaction was developed with 3,3-diaminobenzidinetetrahydrochloride (Sigma Chemical Co., St. Louis, Mo., USA). Thesections were evaluated blindly by counting the labeled cells intriplicates in 10 high-power fields.

Western blots. Protein was extracted from liver tissue samples withPBSTDS buffer (50 mM Tris, 150 mM NaCl, 0.1% SDS, 1% sodiumdeoxycholate, and 1% Triton X-100, pH 7.2). Proteins (30 μμg/sample) inSDS-loading buffer (50 mM Tris, pH 7.6, 10% glycerol, 1% SDS) weresubjected to 12% SDS-PAGE and transferred to nitrocellulose membrane(Bio-Rad Laboratories Inc., Hercules, Calif., USA). The gel was thenstained with Coomassie blue to document equal protein loading. Themembrane was blocked with 3% dry milk and 0.1% Tween-20 (Amersham,Arlington Heights, Ill., USA) in PBS and incubated with rabbit anti-ratHO-I polyclonal Ab (Sangstat Corp., San Francisco, Calif., USA). Thefilters were washed and then incubated with horseradish peroxidasedonkey anti-rabbit Ab (Amersham Life Sciences, Arlington Heights, Ill.,USA). Relative quantities of HO-I protein were determined using adensitometer (Kodak Digital Science 1D Analysis Software, Rochester,N.Y., USA) and results were expressed in absorbance units (AU).

Statistics. Results are expressed as mean±SEM. We used the Tukey-Fisherleast-significant difference (LSD) criterion for judging statisticalsignificance where P values of less than 0.05 were consideredstatistically significant.

Results

HO-I overexpression prolongs OLT survival and improves hepatic function.This experiment examined whether exogenous manipulation of HO-Iexpression could extend the survival of liver transplants. OLTs wereperformed using steatotic Zucker livers that were cold stored for 4hours before transplant into syngeneic lean Zucker rats. The treatmentgroups received a single dose of CoPP or Ad-HO-I gene transfer 24 hoursbefore liver procurement. As shown in FIG. 6, recipients of liverisografts that were stored before transplantation in UW solution alonehad a 40% survival rate at 14 days (4 out of 10). In contrast,recipients of liver isografts pretreated with CoPP showed 80% survivalrate (8 out of 10). Livers pretreated with Ad-HO-I had 81.8% survivalrate at 2 weeks (9 out of 11). Indeed, 8 out of 10 lean Zucker ratsengrafted with livers from CoPP-treated obese Zucker donors were stillalive at well over 100 days after transplant. Prolonged survival afterCoPP or Ad-HO-I pretreatment correlated with improved OLT function asevidenced by sGOT levels. Hence, at day 1, 7, and 14 posttransplant,sGOT levels (1 U/L) in control untreated OLTs of 2695, 1570, and 460,respectively, were significantly higher as compared with correspondingCoPP-pretreated (1838, 477, and 198, respectively; P<0.05) orAd-HO-I-pretreated (1628, 244, and 137, respectively; P<0.05) OLTs.

Liver histology and MNC infiltration in the OLT model. Hepatocyte damagein the OLT model was assessed by a modified Suzuki's classification, asdescribed above. At day 1 after transplant, control untreated liverisografts showed severe disruption of lobular architecture by ballooningchange, significant edema around portal areas, and moderate to severebile duct proliferation (Suzuki score=3.33±0.58). In addition, moderateneutrophil infiltration and hepatocyte necrosis with extreme pallor thatsignifies glycogen depletion in the damaged hepatocytes, were prominentin this OLT group. In contrast, CoPP pretreated liver isografls at day 1showed less neutrophil infiltration and significantly less pallor inaddition to complete preservation of lobular architecture with noevidence of congestion or necrosis (score=1.33±0.70). TheAd-HO-I-pretreated isografts showed much less neutrophil infiltration ascompared with untreated controls; there was no sinusoidal congestion orhepatocyte necrosis and complete preservation of lobular architecture(score 1.50±0.5). Most histologic features characteristic for ischemicpathology resolved by 14-100 days in those 40% of untreated OLTrecipients that survived 2 weeks. However, unlike in theCoPP/Ad-HO-I-pretreated groups, untreated controls still showedsignificant bile duct proliferation.

Liver isografts from untreated obese Zucker donors showed massive MNCinfiltration as early as at 24 hours (T cells: 9±3;monocytes/macrophages: 136±31). In contrast, Zucker rats pretreated withCoPP revealed significantly decreased numbers of intragraft MNC by day 1(T cells: 2±1; monocytes/macrophages 71±12; P<0.03 and P<0.05,respectively). We found some heterogeneity in long-term liver graftsharvested at day 100. Thus, about 50% of untreated grafts showed denseinfiltration by T cells and monocytes/macrophages, followed by severehepatocellular injury; the remainder were characterized by moderate MNCinfiltration and largely preserved hepatocyte architecture. In contrast,all grafis in the CoPP group showed good preservation of hepatocytearchitecture and only mild MNC infiltrate.

Western, analysis of HO-I expression in the OLT model. Western blotswere employed to correlate histologic findings with local HO-Iexpression in liver isografts. The relative expression levels inabsorbance units were analyzed by densitometer. Improved hepaticfunction after CoPP treatment was accompanied by enhanced HO-Iexpression at day 1, 7, 14, and 100 after transplant (1.21-2.14 AU). Incontrast, the corresponding liver isografts from untreated Zucker ratsshowed little HO-I expression (0.09-0.85 AU).

HO-I overexpression, as documented by Western blot analysis, improvedliver function, preserved hepatocyte integrity, and decreasedinflammatory MNC infiltration, with resultant prolongation of survivalafter transplantation. Exogenous upregulation of HO-I prevented orsignificantly decreased hepatic injury in a clinically relevant andwell-defined ex vivo rat fatty liver model of syngeneic OLT. EnhancedHO-I expression improved animal survival from 40% in untreated controlsto about 80% after CoPP treatment or local Ad-HO-I gene delivery, anultimate test for the liver function. Collectively, these results areconsistent with the ability of HO-I to protect cells from oxidativeinjury.

Example 4 Heme Oxygenase-I Protection from Cold Eschemia/Reperfusion(I/R) Injury

Materials and Methods

Animals, metaloporphorins and adenovirus constructs are described inExample 3.

Ex vivo cold ischemia model. Genetically obese Zucker rats underwentether anesthesia and systemic heparinization. After skeletonization ofthe liver, the portal vein, bile duct, and inferior vena cava werecannulated, and the liver was flushed with 10 mL of University ofWisconsin (UW) solution. Control livers from untreated obese Zucker ratswere stored for 6 hours at 4° C. in UW solution (n=6). There were 4treatment groups. Group 1 animals received CoPP, the HO-I inducer (5mg/kg intraperitoneally) 24 hours before liver harvest (n=6). Group 2rats were infused with Ad-HO-I or Ad-β Gal (2.4×10⁹ plaque-forming units[pfu] intraperitoneally) 24 to 48 hours before the procurement (n=4-10).Group 3 donors were treated with Ad-HO-I (2.4×10⁹ pfu intravenously) atday −2, followed 1 day later by infusion of ZnPP (20 mg/kgintraperitoneally), the HO-I inhibitor (n=4). Group 4 rats received ZnPPalone (20 mg/kg intraperitoneally) at 24 hours before harvest (n=4). Alllivers were procured at day 0, stored for 6 hours at 4° C. in UWsolution, and then perfused on an isolated perfusion rat liverapparatus, as described. The Zucker livers were perfused ex vivo for 2hours while temperature, pH, and inflow pressure were kept constant.Portal vein blood flow and pressure were recorded every 15 minutes,whereas bile output was monitored every 30 minutes. Portal vein bloodflow was adjusted to maintain portal pressures of 13 to 18 cmH₂O. Bloodwas collected at 30-minute intervals and serum glutamic-oxaloacetictrans-aminase (sGOT) levels were measured using an autoanalyzer fromANTECH Diagnostics (Irvine, Calif., USA). Following 2 hours ofperfusion, a portion of the liver was snap-frozen for mRNA extractionand Western blot analysis of HO-I expression; the remaining tissuesamples were fixed in formalin for hematoxylin and eosin (H&E) staining.

Hisiology and immunohistochemistry. Liver specimens were fixed in a 10%buffered formalin solution and embedded in paraffin. Sections were madeat 4 μμm and stained with H&E. The histologic severity of I/R injury inthe ex vivo perfusion model was graded using International BanffCriteria. Using these criteria, lobular disarray and ballooning changesare graded from 1 to 4, where no change is given a score of 1 and severedisarray or ballooning changes is given a score of 4.

Western blots were performed as described in Example 3.

Statistics. Results are expressed as mean±SEM. Statistical comparisonsbetween the groups in the ex-vivo perfusion model were performed usingrepeated measure ANOVA. We used the Tukey-Fisher least-significantdifference (LSD) criterion for judging statistical significance where Pvalues of less than 0.05 were considered statistically significant.

Results

The effects of HO-I-inducing agents in the ex vivo steatotic rat livercold ischemia model followed by reperfusion. To test that overexpressionof HO-I decreases I/R-mediated hepatocyte injury, we monitored portalvein blood flow, bile production, and sGOT levels in livers from obeseZucker rats that were either untreated or pretreated with HO-I-inducingagents and then perfused for 2 hours on the isolated perfusion rat liverapparatus.

Pretreatment of Zucker rats with synthetic metalloporphyrin CoPP orAd-HO-I gene transfer exerted equally protective effects against liverI/R injury. Both modalities significantly improved portal blood flowthroughout the 2-hour reperfusion period, as compared with untreatedcontrols (P=0.0001). In addition, both CoPP and Ad-HO-I significantlyincreased bile production (P<0.05), as compared with controls. TheI/R-induced hepatocyte injury measured by sGOT release was also markedlyreduced in the CoPP/Ad-HO-I treatment groups as compared with controls.For instance, at 60 minutes of reperfusion, sGOT concentrations were53.3±8.23 IU/L and 68.8±10.1 IU/L in the CoPP and Ad-HO-I groups,respectively, versus 102±8.23 IU/L in untreated controls (P<0.02). Incontrast, Ad-β Gal gene transfer did not affect the extent of I/R insultsuffered otherwise by steatotic rat livers.

ZnPP abrogates the beneficial effects of HO-I upon hepatic I/R injury.To determine if the amelioration of hepatocyte injury in this I/R modelwas indeed mediated by an increase in HO-I activity, prospective liverdonors were pretreated with ZnPP, a potent HO-I inhibitor. Unlike in theCoPP group, livers procured from obese Zucker rats pretreated with ZnPPalone exhibited diminished portal blood flow and bile production,effects that were accompanied by augmented hepatocyte injury comparablewith otherwise untreated fatty controls. Interestingly, infusion of ZnPPabolished Ad-HO-I-mediated protective effects upon I/R injury insteatotic rat livers. Therefore, portal blood flow and bile productionwere significantly (P<0.05) decreased, and hepatocyte function becameimpaired after adjunctive ZnPP treatment, as compared with Ad-HO-Imonotherapy.

Liver histology, in the ex vivo cold ischemia model followed byreperfusion. The I/R-induced hepatocyte injury in the ex vivo model wasgraded using Banff's criteria. In the untreated fatty Zucker group,there was severe disruption of lobular architecture with marked zone 3ballooning change, focally associated with hepatocyte necrosis (Banff'sscore=3.0±0.63). The ZnPP-treated livers showed somewhat less lobularballooning changes, but more sinusoidal and vascular congestion(score=2.86±0.12). In marked contrast, CoPP-treated livers showedcomplete preservation of the lobular architecture with no signs ofhepatocyte necrosis (score=1.21±0.39). Similarly, livers transduced withAd-HO-I revealed focal areas of mild vacuolar degeneration with minimalhepatocyte necrosis (score=1.68±0.51). However, livers procured fromanimals treated with Ad-HO-I plus ZnPP were characterized by severedisruption of the lobular architecture, similar to the control untreatedgroup, with profound zone 3 ballooning change accompanied by confluentareas of hepatocyte necrosis (score=2.74±0.26). Livers treated with Ad-βGal revealed less necrosis compared with the untreated group, but hadsevere architectural disruption and vascular congestion(score=3.0±1.41).

Western analysis of HO-I expression in tie ex vivo I/R injury model.Western analysis was used to evaluate HO-I expression in liver samplesfollowing cold ischemia at the completion of 2-hour perfusion period.The relative expression levels of HO-I protein in AU were analyzed bydensitometer. Preservation of hepatic function after CoPP pretreatmentor Ad-HO-I gene transfer was accompanied by enhanced HO-I expression(2.46 and 2.12 AU, respectively). In contrast, HO-I was diminished afteradjunctive ZnPP infusion (1.18 AU) and virtually undetectable inuntreated (0.11 AU) and ZnPP-pretreated (0.12 AU) controls.

To test that stress-induced upregulation of HO-I reduces I/R insult insteatotic rat livers, we have chosen 2 distinct HO-I-inducingapproaches. First, donor rats were pretreated with CoPP (5 mg/kgintraperitoneally), a regimen that increases HO-I protein levels in ratlivers by 250% in a rat sandwich ELISA. Second, because infusion of CoPPin high doses may modulate other heme enzymes such as nitric oxidesynthase (NOS) and guanylate cyclase, we have also used Ad-based genedelivery to provide “proof of principle” and to selectively upregulateHO-I expression in prospective liver donors. Western blot analysisconfirmed increased HO-I protein expression in the ex vivo I/R modelusing Ad-HO-I-transduced rat steatotic livers.

The beneficial effects in the ex-vivo I/R-injury model were reflected bythe ability of exogenously upregulated HO-I to improve portal vein bloodflow, increase bile production, and depress sGOT levels, allwell-accepted parameters of hepatic function. Portal blood flow ismostly affected by resistance in the graft caused by lobular ballooning,hepatocyte swelling, and sinusoidal congestion. In this ex vivoperfusion model, the improved portal venous blood flow represents lesshepatocyte injury and lobular disarray in the liver rather than theendothelium-dependant vasodilatory effects of carbon monoxide.Collectively, these results are consistent with the ability of HO-I toprotect cells from oxidative injury.

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

The invention now being fully described, it will be apparent to one ofordinary skill in the art that many changes and modifications can bemade thereto without departing from the spirit or scope of the appendedclaims.

1. A method for extending the survival of an organ transplant in arecipient, said method comprising: contacting cells of an organtransplant with an adenoviral vector comprising a nucleic acid having atleast 80% sequence identity to nucleotides 81-944 of the human hemeoxygenase-I nucleic acid sequence of SEQ ID NO: 1, wherein said nucleicacid encodes a polypeptide having heme-oxygenase activity; and wherebythe survival time of said organ transplant is extended.
 2. The methodaccording to claim 1, wherein said nucleic acid comprises nucleotides81-944 of the human heme oxygenase-I nucleic acid sequence of SEQ IDNO:
 1. 3. The method according to claim 1, wherein said contacting is exvivo.
 4. The method according to claim 1, wherein said contacting is invivo.
 5. The method according to claim 1, wherein said organ transplantis an allograft.
 6. The method according to claim 5, wherein saidallograft is a heart.
 7. The method according to claim 1, wherein saidcontacting is accomplished by direct injection of said adenoviral vectorinto said organ.
 8. The method according to claim 1, wherein the hemeoxygenase-I activity in said cells is increased.
 9. A method forextending the survival of an organ transplant in a recipient, saidmethod comprising: contacting cells of an organ transplant with anadenoviral vector comprising a nucleic acid encoding a polypeptide withat least 80% amino acid sequence identity with the human hemeoxygenase-I encoded by nucleotides 81-944 of the nucleic acid sequenceof SEQ ID NO: 1, wherein said polypeptide has heme-oxygenase activity,and whereby the survival time of said organ transplant is extended. 10.The method according to claim 9, wherein said polypeptide compriseshuman heme oxygenase encoded by nucleotides 81-944 of the nucleic acidof SEQ ID NO:
 1. 11. The method according to claim 9, wherein the hemeoxygenase-I activity in said cells is increased.
 12. The methodaccording to claim 9, wherein said contacting is ex vivo.
 13. The methodaccording to claim 9, wherein said contacting is in vivo.
 14. The methodaccording to claim 9, wherein said organ transplant is an allograft. 15.The method according to claim 14, wherein said allograft is a heart. 16.The method according to claim 9, wherein said contacting is accomplishedby direct injection of said adenoviral vector into said organ.